Enhanced delivery of immunosuppressive drug compositions for pulmonary delivery

ABSTRACT

The present invention includes compositions and methods for making and using a rapid dissolving, high potency, substantially amorphous nanostructured aggregate for pulmonary delivery of tacrolimus and a stabilizer matrix comprising, optionally, a polymeric or non-polymeric surfactant, a polymeric or non-polymeric saccharide or both, wherein the aggregate comprises a surface area greater than 5 m 2 /g as measured by BET analysis and exhibiting supersaturation for at least 0.5 hours when 11-15-times the aqueous crystalline solubility of tacrolimus is added to simulated lung fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/522,774, filed Mar. 2, 2010 as a national phase application under 35U.S.C. §371 of International Application Serial No. PCT/US2008/050795,filed Jan. 10, 2008, which claims priority to U.S. ProvisionalApplication Ser. No. 60/884,383, filed Jan. 10, 2007, the entirecontents of each of which are incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of pulmonarydelivery, and more particularly, to novel compositions and methods forthe manufacture of immunosuppressive drug compositions for pulmonarydelivery.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with pulmonary delivery.

The treatment of solid organ transplants, especially lung transplants,with the currently available immunosuppressive drugs is limited due topoor penetration into the lung following oral or intravenousadministration, associated with significant adverse effects followinglong term treatment. Despite the development of oral formulations foreach of these drugs, low and variable systemic bioavailability,significant side effects and a narrow therapeutic window has limitedtheir use and effectiveness.

Pulmonary formulations of cyclosporine have been developed by dissolvingcyclosporine in the solvents such as ethanol or propylene glycol.However, the results with these solvents were unsatisfactory due to theirritating properties of these solvents. Lidocaine anesthesia of airwayswas required prior to aerosol dosing. More importantly, nebulization wascomplicated due to the precipitation of cyclosporine within thenebulization chamber. Furthermore, cyclosporine is highly toxic and hasnot had a significant effect on increasing long-term transplantsurvival. Given the enhanced immunosuppressive activity of tacrolimus(10 to 100 times more potent than cyclosporine), it is clear thatimprovements in delivery of tacrolimus without using irritating solventswill lead to lower infection rates using prophylaxis treatment andlowered cost with more efficacious therapy.

SUMMARY OF THE INVENTION

The present invention relates to the improved pharmaceuticalcompositions that include rapidly dissolving nanoparticles of tacrolimusthat are administered by pulmonary delivery. More particularly, thecompositions made by fast freezing technology are suitable as arespirable nanoparticle with rapid dissolution rate, high surface area,high potency (50-75% potency) and able to overcome poor bioavailabilityof drugs. The compositions of the present invention are highly porous,amorphous and nanostructured particles with high drug dissolution ratesand high surface area enabling effective treatment of organ rejectiondue to enhanced drug bioavailability. The compositions presentedovercome poor and variable bioavailabilities of drugs effective for bothlocal delivery and systemic delivery in the lung.

More particularly, the present invention includes compositions andmethods for the pulmonary delivery of a rapid dissolving, high potency,substantially amorphous nanostructured aggregate of tacrolimus and astabilizer matrix that includes, optionally, a polymeric ornon-polymeric surfactant, a polymeric or non-polymeric saccharide orboth, wherein the aggregate has a surface area greater than 5 m²/g asmeasured by BET analysis and exhibiting supersaturation for at least 0.5hours when 11 to 15-times the aqueous crystalline solubility oftacrolimus is added to simulated lung fluid. The simulated lung fluidmay include 100 mL of modified simulated lung fluid with 0.02% w/vL-α-phosphatidylcholine dipalmitoyl (DPPC) maintained at 37° C. andstirred with a paddle speed of 100 RPM in a small volume dissolutionapparatus. The composition of the present invention will generallyexhibit greater than about 80% drug dissolved in about 1 hour when anamount equivalent to about 0.59-times the aqueous crystalline solubilityof tacrolimus is added to 100 mL of modified simulated lung fluid with0.02% w/v DPPC maintained at 37° C. and stirred with a paddle speed of50 RPM in a small volume dissolution apparatus.

For example, the supersaturation of tacrolimus may be for at least 1, 2,3 or 4 hrs and the nanostructured aggregate may display a solubilitygreater than crystalline solubility in modified simulated lung fluidwith 0.02% w/v DPPC maintained at 37° C. and stirred with a paddle speedof 100 RPM in a small volume dissolution apparatus. The aggregate willgenerally provide a lung deposition of greater than about 0.10 μg/g wetwhole lung tissue weight when administered by nebulization to a mouseweighing between 16 g and 32 g using the pre-clinical rodent dosingapparatus. In one example, the nanostructured aggregate has a surfacearea of greater than about 5, 10, 20 or 30 m²/g. The nanostructuredaggregate may also be provided for immediate release, extended release,pulsed release, delayed release, controlled release and combinationsthereof.

In one example, the composition may be formulated as a dispersion fornebulization that is prepared by admixing the nanostructured aggregatecontaining tacrolimus with an aqueous carrier and nebulized by anebulizer, an air jet nebulizer, an ultrasonic nebulizer or a micro-pumpnebulizer. The respirable fraction of the nebulized droplets isgenerally greater than about 40, 50, 60, 70, or 80% as measured by anon-viable 8-stage cascade impactor at an air flow rate of 28.3 L/min.The composition may be suitably adapted for delivery using a metereddose delivery device a dry powder inhalation device or a pressurizedmetered dose inhalation device.

The substantially amorphous nanostructured aggregate may be made by oneor more of the following methods: freezing spray, freezing into liquid,spray freezing into vapor, ultra-rapid freezing or spray drying. Forexample, the substantially amorphous nanostructured aggregate is made bysolvent precipitation, antisolvent precipitation, continuousprecipitation or evaporative precipitation into aqueous solution. Inanother method, the tacrolimus is dissolved in solvent or co-solventmixture capable of dissolving all of the components of the compositiontogether with a stabilizing pharmaceutical excipient, wherein aresultant dry powder having tacrolimus present in individual particlesat from 5% to 99% by weight is produced upon spray freezing into liquidor ultra-rapid freezing, followed by lyophilization. The tacrolimus maybe combined with any stabilizing pharmaceutical excipient, e.g., acarbohydrate, organic salt, amino acid, peptide, or protein whichproduces a powder upon spray freezing into liquid or ultra-rapidfreezing. Non-limiting examples of stabilizing pharmaceutical excipientsinclude a carbohydrate selected from the group consisting of mannitol,raffinose, lactose, maltodextrin, trehalose and combinations thereof.The aggregate may include one or more highly wettable nanoparticledomains and/or nanostructured aggregates that quickly wet and dissolvein an aqueous solution.

The present invention also includes a method of making an pulmonarycomposition by mixing tacrolimus with a surfactant, a stabilizer, or acombination or a surfactant and stabilizer matrix and ultra-rapidfreezing the tacrolimus and the surfactant/stabilizer matrix into arapid dissolving high potency amorphous nanoparticle by spray freezinginto liquid or ultra-rapid freezing, wherein the nanoparticle has asurface area greater than 5 m²/g as measured by BET analysis andexhibiting supersaturation for at least 0.5 hours when 15-times theaqueous crystalline solubility of tacrolimus is added to modifiedsimulated lung fluid with 0.02% w/v DPPC. For example, the aggregate ofthe present invention displays a solubility of greater than about 2times that of crystalline tacrolimus solubility. The pulmonarycomposition may be provided for immediate release, extended release,pulsed release, delayed release, controlled release and combinationsthereof. The tacrolimus may be dissolved in a solvent together with astabilizing pharmaceutical excipient, wherein a dry powder havingtacrolimus present in individual particles at from 5% to 99% by weightis produced upon spray freezing into liquid or ultra-rapid freezing.

Yet another embodiment of the present invention is a high surface areananoparticle that is an amorphous aggregate tacrolimus nanoparticlewithin a surfactant/stabilizer matrix adapted for pulmonaryadministration with a surface area greater than 5 m²/g as measured byBET analysis and exhibiting supersaturation for at least 0.5 hours when15-times the aqueous crystalline solubility of tacrolimus is added tomodified simulated lung fluid with 0.02% w/v DPPC.

Another embodiment of the present invention includes compositions andmethods for reducing transplant rejection in a subject by mixingtacrolimus with a surfactant, a stabilizer matrix, or a combination or asurfactant and stabilizer matrix; and ultra-rapid freezing thetacrolimus and the surfactant/stabilizer matrix into a rapid dissolvinghigh potency amorphous nanoparticle by spray freezing into liquid orultra-rapid freezing to form a tacrolimus nanoparticle, wherein thetacrolimus nanoparticle comprises a surface area greater than 5 m²/g asmeasured by BET analysis and exhibiting supersaturation for at least 0.5hours when 11 to 15-times the aqueous crystalline solubility oftacrolimus is added to modified simulated lung fluid; and treating thesubject with an effective amount of the tacrolimus nanoparticlecomposition to prevent transplant rejection. In one aspect, thetacrolimus nanoparticle is adapted for pulmonary delivery. In anotheraspect, the tacrolimus nanoparticle is provided to prevent rejection ofa lung transplant. In another aspect, the tacrolimus nanoparticle isprovided at between 0.1 mg/ml to 100 mg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A and 1B are SEM images of nanoparticles that include tacrolimus(TAC):Lactose (1:1) at two different magnifications;

FIGS. 2A and 2B are SEM images of nanoparticles containing tacrolimus(TAC): Poloxamer 407 (4:1);

FIG. 3 is an SEM of TAC Crystalline Bulk Powders;

FIG. 4 shows the X-ray diffraction profiles of TAC URF formulationscompared to unprocessed TAC;

FIG. 5 is a graph that shows dissolution testing results. Dissolutiontesting was conducted at sink conditions using a USP Type 2 apparatus(VanKel VK6010 Dissolution Tester with a Vanderkamp VK650Aheater/circulator); dissolution media was 900 mL of modified simulatedlung fluid containing 0.02% DPPC maintained at 37° C. and stirred at 100rpm; dissolution profiles were determined in replicates of 6;

FIG. 6 is a graph that shows the sink dissolution profiles for (▪)Amorphous URF composition TAC: lactose (1:1), (▴) Crystalline URFcomposition TAC alone and () Unprocessed crystalline TAC. Thedissolution media was modified simulated lung fluid containing 0.02%DPPC at 100 rpm and 37° C. (equilibrium solubility of TAC in this media˜6.8 μg/mL). Dissolution profiles were determined in replicates of 3;

FIG. 7 is a graph that shows the supersaturated dissolution profiles for( ) Amorphous URF composition TAC: lactose (1:1); (▪) Crystalline URFcomposition TAC alone and (---) Equilibrium solubility of TAC in thedissolution media (6.8 μg/mL). The dissolution media was modifiedsimulated lung fluids (SLF) containing 0.02% DPPC at 100 rpm and 37° C.Dissolution profiles were determined in replicates of 3; C is measuredconcentration of TAC at a given time point and Ceq is equilibriumconcentration of TAC;

FIG. 8 is a graph that shows a comparison of mean lung concentration (μgTAC/g tissue) versus time profiles in mice of the URF formulations. ( )Amorphous URF composition TAC: lactose (1:1) and (▪) Crystalline URFcomposition TAC alone;

FIG. 9 is a graph that shows a comparison of mean whole-blood TACconcentration profiles of the URF formulations after a single inhalationadministration. ( ) Amorphous URF composition TAC: lactose (1:1) and (▪)Crystalline URF composition TAC alone;

FIG. 10A: Lung tissue histology from TAC:LAC active group, day 7, FIG.10B: Lung tissue histology from TAC:LAC active group, day 14, FIG. 10C:Lung tissue histology from LAC only control group, day 7, and FIG. 10D:Lung tissue histology from LAC only control group, day 14 (Notes onhistology: a: alveolar spaces; b: capillaries; c: lymph tissue; and d:arterioles with red blood cells present; (20× magnification)).

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, the term “BET Analysis” refers to a method for measuringthe surface area of the exposed surface of a solid sample on themolecular scale. The BET (Brunauer, Emmet, and Teller) theory istypically used to determine the area of solids. Commonly, samples areprepared by heating and simultaneously evacuating or flowing gas overthe sample to remove the liberated impurities. The prepared samples arethen cooled with liquid nitrogen and analyzed by measuring the volume ofgas (typically N₂ or Kr) adsorbed at specific pressures. Krypton gas isoften used when the measured surface is expected to be less than 2 m²/g(typically pharmaceutical samples and natural organic materials). Theskilled artisan will recognize that several types of instruments may beused for measuring surface area, which depends upon the extent of thesurface or the gas required to achieve the best results. Examples ofequipment that may be used includes the ASAP 2020 and ASAP 2405 Kr, theTristar 3000, the Gemini 2380, and the Flowsorb 2310.

As used herein, the term “modified simulated lung fluid with 0.02% w/vDPPC” refers to the composition reported in Table 1 of the referenceDavies, N. M. and Feddah, M. R. (2003) A novel method for assessingdissolution of aerosol inhaler products. International Journal ofPharmaceutics, 255, 175-187. As seen in Table 1 of Davies, et al., thecomposition includes: calcium ion, magnesium ion, potassium ion, sodiumion, bicarbonate, chloride ion, citrate ion, acetate ion, phosphate ion,sulfate ion, and DPPC. The pH ranges between 7.3 and 7.4. The modifiedsimulated lungs fluid as describe herein is composed of calcium,magnesium, potassium, sodium, bicarbonate, chloride, citrate, acetate,phosphate, and sulphate at 5, 2, 4, 145, 156, 31, 114, 1, 7, 2, and 1mEq/L respectively. Additionally, 0.02% L-α-phosphatidylcholine wasadded according to the method described in Davies et al.

TABLE 1 Compositions of actual lung fluid, simulated lung fluids and themodified simulated lung fluid by DPPC (mEq./1). Modified Simulatedsimulated lung lung fluid with Ion Actual^(a) fluid^(b) 0.02% DPPCCalcium, Ca²⁺ 5.0 5.0 5.0 Magnesium, Mg²⁺ 2.0 2.0 2.0 Potassium, K⁺ 4.04.0 4.0 Sodium, Na⁺ 145.0 145.0 145.0 Total cations 156.0 156.0 156.0Bicarbonate, HCO₃ ⁻ 31.0 31.0 31.0 Chloride, Cl⁻ 114.0 114.0 114.0Citrate, H₅C₆O₇ ³⁻ — 1.0 1.0 Acetate, H₃C₂O₂ ⁻ 7.0 7.0 7.0 Phosphate,HPO₄ ²⁻ 2.0 2.0 2.0 Sulphate, SO₄ ²⁻ 1.0 1.0 1.0 Protein 1.0 — —L-α-Phosphati- — — 200_(mg) dylcholine Total anions 156.0 156.0 156.0 pH7.3-7.4 7.3-7.4 7.3-7.4 DPPC: L-α-phosphatidylcholine. ^(a)Diem andLenter (1970). ^(b)Moss (1979).

As used herein, the term “exhibiting supersaturation” refers to asolution that contains more of the dissolved tacrolimus than could bedissolved by the modified simulated lung fluid with 0.02% w/v DPPCcompared to the crystalline tacrolimus.

As used herein, the term “quickly wets” refers to the ability of thenanostructured aggregates to be wetted by the modified simulated lungfluid with 0.02% w/v DPPC or lung fluids in vivo at body temperature(37° C.).

As used herein, the term “wet whole lung tissue weight” refers to thetotal lung weight of a mouse.

As used herein, the term “pharmaceutical carrier” means the inactivecomponents of the pharmaceutical composition.

As used herein, the term “surfactant” means a substance that reduces thesurface tension of a liquid, thereby causing it to spread more readilyon a solid surface. Examples of surfactants for use with the presentinvention, include, all surfactants suitable for administration to thelungs, including sodium salts of cholate, deoxycholate, glycocholte andother bile salts; Span 85, Lauryl-beta-D-maltoside, palmitic acid,glycerol trioleate, linoleic acid, DPPC oleyl alcohol, oleic acid,sodium oleate, and ethyl oleate.

As used herein, the term “pre-clinical rodent dosing apparatus” refersto the apparatus reported by J. T. McConville, P. Sinswat, J. Tam, K. P.Johnston and R. O. Williams III, In vitro and in vivo validation of ahigh-concentration pre-clinical rodent dosing apparatus for inhalation,Proceedings of the American Association of Pharmaceutical Scientists,San Antonio, Tex., October, 2006, relevant portions incorporated hereinby reference. An animal dosing chamber was designed which consisted ofhollow tubing (20×4.5 cm; nominal wall thickness of 0.4 cm) with four1.75 cm adapter holes drilled at 7 cm intervals (2 holes along eachside). The adapter holes were constructed to accept rodent restrainttubes from the Battelle® toxicology testing unit. A standarditraconazole (ITZ) colloidal suspension was nebulized into the apparatusfor 5 minutes using a micro-pump nebulizer. Atomized droplets containingITZ were driven into the chamber at a flow rate of 1 L/min. ITZconcentrations were measured in vitro at the 4 adapter ports, and invivo from the lungs of 8 outbred-ICR mice in the appropriate micerestraining tubes at the adapter ports.

The technology makes use of existing excipients used are all selectedfrom the GRAS list approved by the FDA, their use is approved by theFDA, or they are naturally occurring in mammalian tissues. They are usedfrequently in oral tablet or parenteral preparations and indicateminimal toxicity levels. These are used in conjunction with the activeingredient to form a stable nanosized dosage form.

It is important that processed drug product contains stabilizednanoparticles with high surface area to obtain high dissolution rates.Other processing technologies can be used such as fast freezing,antisolvent and precipitation methods.

It was also found that TAC has the ability to reverse ongoing rejection.Subsequently, numerous studies have confirmed the effectiveness of TACas primary therapy in a variety of solid organ transplants. Ofimportance, the enhanced immunosuppressive activity of TAC is achievedwithout increased risk of infection or malignancy. Although many studiesrevealed that TAC may have superior immunosuppressive activitiescompared to cyclosporine, erratic absorption from the gastrointestinaltract following oral administration have limited the drug's clinicalpotential.

The average oral bioavailability of this drug is approximately 25% inadult patients. High cost of rejection therapy associated withtacrolimus is about $34,200 in the first year. The compositions of thepresent invention composed of porous aggregates of small particles withhigh drug dissolution rates and high surface area enabling effectivetreatment of organ rejection due to enhanced drug bioavailability.

TABLE 2 Surface Area Analysis Specific Surface Area Formulations [m²/g]TAC Bulk Powders 0.533 TAC:Lactose (1:1) 44.28 TAC:Poloxamer 407 (4:1)40.07

Particle Morphology. SEM—The powder samples were sputter coated withgold-palladium for 35 seconds and viewed using a Hitachi S-4500 fieldemission scanning electron microscope. SEM indicates the presence ofporous aggregated small particles

X-Ray Diffraction. The x-ray diffraction pattern of powders wereanalyzed using a Philips 1710 x-ray diffractometer with a copper targetand nickel filter (Philips Electronic Instruments, Inc., Mahwah, N.J.).The leveled powder was measured from 10 to 40 2-theta degrees using astep size of 0.05 2-theta degrees and a dwell time of one second. Nocorresponding tacrolimus peaks can be identified.

Dissolution of oral formulations (n=6). Dissolution testing wasperformed on the powder samples using USP Type 2 apparatus (VanKelVK6010 Dissolution Tester with a Vanderkamp VK650A heater/circulator).An equivalent of 4 mg tacrolimus was added to 900 ml of modifiedsimulated lung fluid with 0.02% DPPC dissolution media and stirred at100 rpm. The dissolution media was maintained at 37.0±0.2° C. Fivemilliliter samples were withdrawn at 10, 20, 30, 60 and 120 minute timepoints, filtered using a 0.45 μm GHP Acrodisc filter and analyzed usinga Shimadzu LC-10 liquid chromatograph (Shimadzu Corporation, Kyoto,Japan). A 70:30 acetonitrile:water mobile phase at 1 ml/min eluted theTAC peak at 6 minutes. For the formulations prepared for FIG. 5, thetacrolimus absorbance was measured at λ=220 nm.

TABLE 3 Anderson Cascade Impactor - Aeroneb ® Professional MicropumpNebulizer data. Total Respirable Emitted Dose fraction MMAD(GSD)Formulations [μg/min] [%] [μm] TAC:Lactose (1:1) 5082 74.6 2.57(2.24)TAC:Poloxamer 407 (4:1) 4917 71.1 2.75(1.86) MMAD = Mass MedianAerodynamic Diameter GSD = Geometric Standard Deviation

Good aerosolization performance was achieved. Greater than 70%respirable fractions are indicated for all formulations using theAeroneb® Professional nebulizer. Mass median aerodynamic diametersbetween 1 and 5 m are also indicated for all formulations, using thisnebulizer.

This study were to produce nanostructured aggregates of tacrolimus (TAC)intended for pulmonary delivery using ultra-rapid freezing (URF), and toinvestigate the physicochemical and pharmacokinetic characteristics ofthe nanostructured aggregates containing amorphous or crystallinenanoparticles of TAC. Two URF formulations were investigated forpulmonary delivery, and compared to bulk unprocessed TAC, these were:TAC and lactose (1:1 ratio; URF-TAC:LAC) and TAC alone (URF-TAC). TACand water soluble excipient i.e. lactose were dissolved in acetonitrileand water, respectively. Two solutions were mixed to obtain 60:40 ratioof the resulting organic/aqueous co-solvent system which was then frozenon the cryogenic substrate. The cosolvent was then frozen on the URFcryogenic substrate and the frozen compositions were collected andlyophilized to form the dry powder for nebulization. In vitro resultsrevealed similar physiochemical properties for both URF formulations.BET analysis showed high surface areas of 29.3 m²/g and 25.9 m²/g forthe URF-TAC:LAC and URF-TAC, respectively, and 0.53 m²/g for theunprocessed TAC, respectively. Scanning electron microscopy (SEM) showednanostructured aggregates containing nanoparticles of TAC. Thedissolution of TAC was 83.6% at 1 hr for the URF-TAC:LAC, compared to80.5% for the URF-TAC and 30% dissolved for the unprocessed TAC,respectively. Similar aerodynamic particle sizes of 2-3 μm, and fineparticle fraction between 70-75% for the URF-TAC:LAC and URF-TAC weredetermined by cascade impactor data. X-ray diffraction (XRD) resultsindicated that URF-TAC was crystalline, whereas URF-TAC:LAC wasamorphous. The supersaturated dissolution profiles were in agreementwith these results. URF-TAC:LAC displayed the ability to supersaturatein the dissolution media to about 11-times crystalline equilibriumsolubility. In vivo studies were conducted in mice by dispersing the URFformulations in deionized water and nebulizing the dispersed URFformulations using a specially designed nose-only dosing apparatus. Thepharmacokinetic profiles obtained showed comparable AUC₍₀₋₂₄₎, higherC_(max), and lower T_(max) for the URF-TAC:LAC compared to the URF-TAC.Therefore, rapidly dissolving, pulmonary formulations containingnanostructured aggregates of amorphous or crystalline TAC were developedusing the URF technology. The URF processed formulations weredemonstrated to be effectively delivered as an aqueous dispersion of TACnanoparticle via nebulization, with a similar in vivo performance bydisplaying the comparable extent of drug absorption.

Tacrolimus (TAC) is a widely used immunosuppressive agent isolated fromStreptomyces tsukubaensis. It has proven to be a potentimmunosuppressant in transplantation medicine for treatment of organrejection and different immunological diseases such as pulmonaryfibrosis and bronchiolar asthma [1-3]. TAC was first introduced asrescue therapy when cyclosporin A (CsA) therapy failed to prevent graftrejection. It has a mechanism of action similar to that of CsA, but itsimmunosuppressive activity is 10- to 100-times more potent than CsA[4,5]. TAC is currently available in both an intravenous and oral dosageform (commercially known as Prograf®). However, these current availabledosage forms of the drug are poorly tolerated and provide a variableand/or low bioavailability [6]. The oral formulations of TAC present aconsiderable challenge as the drugs are practically insoluble in waterand extensively metabolized from both CYP3A4 metabolism andp-glycoprotein efflux transport within the intestinal epithelium[7]. Theoral bioavailability of TAC varies from 4% to 93% [8]. Inefficient orerratic drug absorption is primarily the result of incomplete absorptionfrom the gastrointestinal tract and first-pass metabolism, which issubject to considerable inter-individual variation[8].

This invention focuses on a pulmonary drug delivery system based on,nanoparticles of TAC in order to overcome the above mentioned problemsto improve bioavailability. The appealing aspects of inhaled drugnanoparticles include: Rapid dissolution of nanoparticles in the lungand the avoidance of hepatic first pass metabolism (which is especiallyuseful for a drug that undergoes extensive metabolism in liver) [9,11].Additionally, inhaled nanoparticles can increase local drugconcentrations in the lung for potential therapeutic use in lungtransplantation and pulmonary diseases. The treatment of lung transplantrecipients is often limited due to poor penetration of drug into thelung following oral or intravenous administration[12]. Aerosolized drugwill have direct access to the graft in lung transplant offering thepossibility of much higher drug levels [13]. However, a majordisadvantage of pulmonary delivery for drugs like TAC is limitations inthe levels and types of excipients that are considered safe to use inpulmonary formulations. Although many surfactants or polymers such ascyclodextrins, poloxamers, polyethylene glycols (PEG) and glycerol havebeen studied in pulmonary formulations to aid drug solubilization inmany research studies [14-16], these excipients have not been approvedyet for commercial use by the FDA because of potential toxicity in thelung. Several clinical studies have demonstrated effective pulmonarydelivery of CsA solutions in ethanol or propylene glycol prior toaerosolization in lung transplantation models [17-19]. However, thesolvents have produced the results have shown unsatisfactory due to theirritating properties of these solvents to the airways. In addition, theuse of high levels of ethanol or propylene glycol in formulationsintended for pulmonary delivery have yet to be widely studied in humans.Recently, liposome technology has been investigated as a non-irritatingalternative for pulmonary delivery of CsA, but the formulation had lowdrug loading and thus requires a lengthy nebulization period [20].

Pulmonary formulations containing TAC manufactured by ultra-rapidfreezing (URF), without the inclusion of surfactants or polymericexcipients, were investigated. URF technology is a continuous, scalablecryogenic process produces nanostructured aggregates with high surfacearea resulting in high enhanced drug dissolution rates. Previously,spray freezing into liquid (SFL) was reported [21-26]. The rapidfreezing rates achieved with the SFL process led to the production ofamorphous nanostructured aggregates composed of primary particles,ranging from 100 to 200 nm, with high surface areas, high wettabilityand significantly enhanced dissolution rates. The URF process yieldsparticles with similar properties as those produced by SFL. In URF asolution of the active and excipient in a suitable organic solvent oraqueous co-solvent is applied to the surface of a cryogenic solidsubstrate. The spray is frozen instantaneously, in 50 ms to 1 s, ontothe surface of cryogenic solid substrate in a continuous manner [27,28].URF powders exhibit desirable properties for enhancing bioavailabilitysuch as high surface area, increased drug dissolution rates, andamorphous character.

Nanostructured aggregates composed of amorphous or crystalline primarynanoparticles of TAC produced by the URF process are suitable forpulmonary delivery by nebulization, resulting in high lung and bloodconcentrations. The hypothesis is that high surface area and rapiddissolution rate obtained from nanostructured aggregates of TAC promotehigh systemic drug absorption via the lung, whilst still maintaining adesirable pulmonary residence time for potential local therapy. Relevantphysicochemical properties (e.g. surface area, dissolution,crystallinity) of TAC nanostructured aggregates were characterized inorder to understand how they influence in vivo drug absorption followingsingle-dose nebulization of the particle dispersions.

TAC was kindly provided by The Dow Chemical Company (Midland, Mich.).Lactose, magnesium chloride hexahydrate, sodium chloride, potassiumchloride, sodium phosphate dibasic anhydrous, sodium sulphate anhydrous,calcium chloride dihydrate, sodium acetate trihydrate, sodiumbicarbonate and sodium citrate dihydrate were analytical grade andpurchased from Spectrum Chemicals (Gardena, Calif.).Dipalmitoylphosphatidylcholine (DPPC) was purchased from Sigma-AldrichChemicals (Milwaukee, Wis.). High performance liquid chromatography(HPLC) grade acetonitrile (ACN) was purchased from EM Industries, Inc.(Gibbstown, N.J.). Liquid nitrogen was obtained from Boc Gases (MurrayHill, N.J.). Deionized water was prepared by a Milli-Q purificationsystem from Millipore (Molsheim, France).

Exemplary preparation of URF Formulations. TAC formulations wereprocessed using URF. Two URF formulations considered for pulmonarydelivery were TAC:lactose in a 1:1 ratio (URF-TAC:LAC) and TAC alone(URF-TAC). The compositions were prepared by dissolving TAC andhydrophilic excipient (if any) at a 1:1 ratio and 0.75% solids in a60/40 mixture of acetonitrile and water. The solution of drug wasapplied to the surface of solid substrate, which is cooled using acryogenic substrate maintained at −50° C. The frozen compositions werethen collected and the solvent was removed by lyophilization usingVirTis Advantage Tray Lyophilizer (VirTis Company Inc., Gardiner, N.Y.).The lyophilization recipes used in this study is outlined in Appendix A.The dried powders were stored at room temperature under vacuum.

In Vitro Characterization of Powders for Pulmonary. X-ray PowderDiffraction (XRD). The XRD patterns of the powders were analyzed using aPhilips 1710 x-ray diffractometer with a copper target and nickel filter(Philips Electronic Instruments, Inc., Mahwah, N.J.). Each sample wasmeasured from 5 to 45 2θ degrees using a step size of 0.05 2θ degreesand a dwell time of one second.

BET Specific Surface Area Analysis. Specific surface area was measuredusing a Nova 2000 v.6.11 instrument (Quantachrome Instruments, BoyntonBeach, Fla.). A known weight of powder was added to a 12 mm Quantachromebulb sample cell and degassed for a minimum of three hours. The datarecorded were then analyzed according to BET theory using NOVA EnhancedData Reduction Software v. 2.13.

Scanning Electron Microscopy (SEM). A Hitachi S-4500 field emissionscanning electron microscope (Hitachi High-Technologies Corp., Tokyo,Japan) was used to obtain SEM micrographs of the powder samples. Sampleswere mounted on conductive tape and sputter coated using a model K575sputter coater (Emitech Products, Inc., Houston, Tex., USA) withgold/palladium for 30 s. An accelerating voltage of 5-15 kV was used toview the images.

Dissolution Testing at Below Equilibrium Solubility. Dissolution testingat below equilibrium solubility was performed on the URF powder samplesusing a United States Pharmacopeia (USP) 27 Type 2 dissolution apparatus(VanKel VK6010 Dissolution Tester with a Vanderkamp VK650Aheater/circulator, Varian, Inc. Palo Alto, Calif.). Powder samples (0.4mg of TAC) equivalent to approximately 59% of the equilibrium solubility(6.8 μg/mL) were added to 100 mL of modified simulated lung fluids (SLF)with 0.02% DPPC as dissolution media [29]. The dissolution media wasmaintained at 37.0±0.2° C. and stirred at a constant rate of 50 rpm.Samples (1 mL) were withdrawn at 10, 20, 30, 60 and 120 minute timepoints, filtered using a 0.45 μm GHP Acrodisc filter (VWR, Inc., WestChester, Pa.) and analyzed using a Shimadzu LC-10 liquid chromatograph(Shimadzu Corporation, Kyoto, Japan) equipped with an Alltech ODS-2, 5μm C₁₈ column (Alltech Associates, Inc., Deerfield, Ill.). The mobilephase consisted of a 70:30 (v/v) ACN: Water mixture, used at a flow rateof 1 mL/min. The maximum absorbance was measured at wavelengthλ_(max)=214 nm.

Dissolution Behavior in the Formation of Supersaturated Solutions.Supersaturated dissolution profiles were generated according to themethod previously described except using the small volume dissolutionapparatus equipped with a paddle stirring mechanism. Each drugformulation was weighed out which corresponded to approximately 15-timesthe aqueous crystalline solubility of TAC in 100 mL of the modifiedsimulated lung fluid with 0.02% DPPC. Paddle speed and bath temperaturewere maintained at 100 rpm and 37° C., respectively. An aliquot (1 mL)were removed from the small volume dissolution vessel at 10, 20, 30 and60 minutes, then at 2, 4 and 24 hours. Each aliquot was filtered througha 0.2 μm nylon filter, and a 0.5 mL aliquot of each filtered solutionwas immediately mixed with 1 mL of acetonitrile (to ensure nore-crystallization of drug previously dissolved at 37° C.). The sampleswere analyzed for TAC concentration using the same HPLC proceduredescribed previously. All studies were performed in triplicate.

In Vitro Aerosol Performance. The in vitro deposition characteristics ofthe dispersed and nebulized TAC formulations were investigated using anon-viable 8-stage cascade impactor (Thermo-Electron Corp., Symrna, Ga.,USA). The aerosolization behavior was described in terms of totalemitted dose (TED), fine particle fractions (FPFs), mass medianaerodynamic diameters (MMAD) and geometric standard deviation (GSD). Thecascade impactor was assembled and operated in accordance with USPmethod 601 to assess the drug delivered. The powders were dispersed inwater (10 mg/mL) and nebulized using an Aeroneb® Professional micropumpnebulizer (Nektar Inc., San Carlos, Calif.) for 10 minutes at an airflow rate of 28.3 L/min. The flow rate was maintained by a vacuum pump(Emerson Electric Co., St. Louis, Mo., USA) and calibrated by a TSI massflow meter (Model 4000, TSI Inc., St. Paul, Minn., USA). The massdeposited on each of the stages was collected and analyzed by HPLC asdescribed herein. Each study was repeated in triplicate.

In Vivo Mouse Studies. Pulmonary Administration of URF Formulations.Pulmonary dosing of URF formulations was performed in healthy male ICRmice (Harlan Sprague Dawley, Inc., Indianapolis, Ind.). The studyprotocol was approved by the Institutional Animal Care and Use Committee(IACUCs) at the University of Texas at Austin, and all animals weremaintained in accordance with the American Association for Accreditationof Laboratory Animal Care. Mice were acclimated and pre-conditioned inthe restraint tube (Battelle, Inc., Columbus, Ohio) for 10-15 min./dayfor at least 2 days prior to dosing. Proper pre-conditioning isessential for reducing stress to mice, and maintaining a uniformrespiration rate for the animals. A small animal dosing apparatus forinhalation was used to dose the mice for this study. The dosingapparatus includes a small volume hollow tube with dimensions of 20×4.5cm (nominal wall thickness of 0.4 cm) with four 1.75 cm adapter holesdrilled at 7 cm intervals (2 holes along each side). The adapter holeswere constructed to accept rodent restraint tubes from the Battelletoxicology testing unit.

The URF processed powders were re-dispersed in water (10 mg/mL) followedby sonication for 1 min. prior to dosing. Nebulization of 3 mL ofdispersions was conducted using an Aeroneb Professional micropumpnebulizer for 10 min. dosing period. After pulmonary dosing, the micewere removed from the dosing apparatus and rested for 15 min. Two micewere sacrificed at each time point by CO₂ narcosis (0.5, 1, 2, 3, 6, 12,24 and 48 hours). Whole blood (1-mL aliquots) was obtained via cardiacpuncture and analyzed according to the standard ELISA procedure outlinedhereinbelow. In addition, necropsy was performed on each mouse toextract lung tissue. Samples were stored at −20 C until assayed. TACconcentrations in lung tissue were determined using a previously HPLCassay as described below.

Enzyme-Linked Immunosorbent Assay (ELISA) for Analysis of TACConcentrations in Blood. The determination of TAC in whole blood wasperformed using the PRO-Trac™ II FK 506 ELISA assay kit (Diasorin Inc.,Stillwater, USA) in accordance with the manufacturer's instructions.Specifically, 50 μL of whole blood sample or standards were placed intoa conical 1.5 mL polypropylene tube. Digestion reagent was freshlyreconstituted, and 300 μL was added to all tubes. The tubes werevortexed for 30 seconds and incubated at room temperature for 15 min.These tubes was then placed on an aluminum heating block circulated with75° C. water bath for 15 min to stop proteolysis. After vortexing, thetubes were centrifuged at room temperature at 1,800 xg for 10 min. Thesupernatant (100 μL) was transferred to microtiter plate wells induplicate from each centrifuged tube. Capture monoclonal anti-FK506 (50μL) was added to the each well, and the plate was shaken at roomtemperature at 700 rpm for 30 min. TAC horseradish peroxidase conjugate(504) was then added to each well, and the plate was shaken at roomtemperature at 700 rpm for an additional 60 min. The plate was washed,before the addition of 200 μL chromogen. The plate was then shaken at700 rpm for a further 15 min at room temperature. The subsequentreaction in each plate well was terminated by the addition of 100 μL ofstop solution. The absorbance in each well was read at the dualwavelengths of 450 and 630 nm. Data was plotted according to afour-parameter logistic (4PL) curve-fitting program.

Solid Phase Extraction and Drug Analysis of Lung Tissues using HPLC.Lung extraction was carried out using solid phase extraction to obtainTAC levels using reverse phase HPLC. The total lung weight was recordedindividually from each mouse. Lung tissues were homogenized using aPolytron rotor-stator homogenizer (VWR Scientific Corporation, WestChester, Pa.) for 40 seconds in 1 mL of normal saline. The homogenizedlung samples were then mixed with 0.5 mL solution of 0.4 N zinc sulfateheptahydrate in the mixture of methanol/water (70:30) solution andvortex mixed for 30 seconds. Acetonitrile (1 mL) was added to thehomogenized samples before a further vortex mixing for 1.5 minutes,followed by centrifugation at 3000 rpm for 15 minutes to obtain a clearsupernatant. Next, the supernatant was collected into a clean vialcontaining 1 mL purified water. Meanwhile C18 cartridges for solid phaseextraction (Supelco Inc., Bellefonte, Pa.) were preconditioned. First,these columns were pretreated with 2 mL of acetonitrile, followed by 1mL methanol and then washed with 1 mL of water before loading thesupernatant through the column.

The sample was transferred and drawn slowly through the column byreducing the vacuum. The column was washed again by passing 1.5 mLmixture of methanol/water (70:30) solution, followed by 0.5 mL ofn-hexane and allowed it to dry under vacuum. The sample was finallyeluted with 2 mL of acetonitrile (0.5 mL×4). The eluted material wasevaporated under a dry nitrogen stream and then reconstituted with 250μL of mobile phase using the previously described HPLC assay (below).Data was expressed as μg TAC/gram wet lung tissue analyzed.

Pharmacokinetics and Statistical Analysis. The lung tissue concentrationvs. time was investigated using a non-compartmental model, while thewhole blood concentration vs. time was evaluated using one-compartmentalanalysis from extravascular administration (via the lung compartment).Pharmacokinetic parameters were calculated using WinNonlin version 4.1(Pharsight Corporation, Mountain View, Calif.). The pharmacokineticprofile of TAC was characterized by maximum concentration (C_(max)),time to C. (T_(max)), half-life (T_(1/2)) and area-under-the-curve (AUC)between 0-24 hours. AUC was calculated using the trapezoidal rule;C_(max) and T_(max) were determined from the concentration-timeprofiles; T_(1/2) was calculated by using the elimination rate constant(K_(el)); K_(el) was obtained from the ln concentration-time profiles.

In vitro characterization of URF formulations. The physicochemicalproperties of TAC powders produced by URF were investigated and comparedto the unprocessed TAC. The XRD patterns of the URF formulations andunprocessed TAC are shown in FIG. 4. The diffractogram of URF-TAC wassimilar to that of unprocessed TAC, indicating a high degree ofcrystallinity. However, the XRD pattern of URF-TAC:LAC confirmed thatthis composition was amorphous. This suggests that lactose inhibitedcrystallization of TAC. It is well known that sugars such as lactose canbe used to stabilize amorphous drugs, peptides and proteins duringdrying and subsequent storage [30,31]. The addition of sugars has beenshown to extend the shelf life of amorphous systems by preventingcrystallization. In addition, lactose is generally regarded as safe(GRAS) for use as an excipient in inhalation systems [32]. This is dueto its non-toxic and degradable properties after administration [33].

SEM micrographs of the two URF processed formulations are shown in FIG.5 reveal distinct differences in morphology. The morphology ofURF-TAC:LAC (FIG. 5 a-5 b) showed highly porous, nanostructuredaggregates. The micrograph at high magnification in FIG. 5 b revealedthat the aggregates were composed of branched interconnected nanorodswith a diameter of approximately 100-200 nm. URF-TAC (FIG. 5 c-5 d)appeared as more dense aggregates composed of submicron primaryparticles. In contrast, the SEM micrograph of unprocessed TAC indicatedan irregular, dense and large crystal plate measuring between 50-100 μmin size (FIG. 5 e). Accordingly, the surface areas obtained by the URFprocessed formulations (URF-TAC:LAC and URF-TAC was 25.9 and 29.3 m²/g,respectively) were significantly higher than (p<0.05) that of theunprocessed drug (0.53 m²/g). This result is corroborated by the porousnanostructured aggregates of the URF powders observed by SEM.

The in vitro aerosol performance measured by cascade impaction foraqueous dispersions prepared from the URF processed powders arepresented in Table 4.

TABLE 4 Physicochemical properties of TAC powder compositions preparedby the URF process and aerosol characteristics of aqueous dispersions ofURF powder compositions delivery by nebulization. Physical Surface TEDState Area (μg/ % MMAD Formulations of Drug (m²/g) min) FPF (μm) GSDURF-TAC:LAC Amorphous 29.3 5082 74.6 2.57 2.24 URF-TAC Crystalline 25.94823 70.2 2.86 1.97 TED: total emitted dose. MMAD: mass medianaerodynamic diameters. GSD: geometric standard deviation. FPF: fineparticle fraction, as percentage of total loaded dose <4.7 μm.

Comparison of the data suggests similar aerodynamic properties of thedrug particles aerosolized from the two URF formulations. The MMAD was2.86 and 2.57 μm for URF-TAC:LAC and URF-TAC, respectively, and the GSDwas less than 2.2 (Table 3). It can be concluded that the aerosoldroplets contain aggregates of nanoparticles that are in the respirablerange by nebulization. Aerodynamic particle size is the most importantparameter in determining drug deposition in the lungs and must beconsidered when developing formulations for pulmonary delivery [34].Aerosolized particles or droplets with a MMAD ranging from 1 to 5 μm aresuitable for deep lung deposition, at the site of the alveoli, wheremaximum absorption may take place [35]. The optimal aerosolizationproperties of both URF formulations are also reflected in the high % FPFranging from 70% to 75%, illustrating efficient lung delivery of drugparticles. The TED was only slightly higher for URF-TAC:LAC (5082μg/min) compared to that of URF-TAC (4823 μg/min). These values were notsignificantly different (p>0.05).

The in vitro dissolution profiles of TAC in SLF media under sinkconditions are shown in FIG. 6. The dissolution rates for both URFprocessed powders were significantly increased (p<0.05) as compared tothe unprocessed TAC. Nanostructured aggregates of the URF processedpowders were able to wet and dissolve quickly upon contact in SLFcontaining 0.02% DPPC, although the formulations contained nosurfactant. For URF-TAC:LAC (i.e., amorphous, nanostructuredaggregates), the dissolution of TAC was 72% in 30 minutes, compared to67% for the URF-TAC (i.e., crystalline nanostructured aggregates) and30% for the unprocessed TAC, respectively. The enhancement is mostlikely attributed to the high porosity and enhanced surface area of URFprocessed powders.

Dissolution of TAC at supersaturated conditions was also conducted inthe same media. Supersaturated dissolution profiles of the URF processedformulations containing 15-times the equilibrium solubility of TAC arecompared in FIG. 7. The concentration obtained for the URF-TAC:LACexceeded the equilibrium solubility of TAC, corresponding to a highdegree of supersaturation in the SLF containing DPPC without thepresence of surfactants or polymers in the formulation. The level ofsupersaturation corresponded to about 11-times the equilibriumsolubility. This was due to the high-energy phase of the amorphous TACparticles. The maximum concentration occurred at 1 hour, and thendecreased to about 3-times equilibrium solubility over the next 4 hours.A supersaturation dissolution profile was not observed for URF-TACbecause of its crystalline nature.

In Vivo Pulmonary Studies. The pharmacokinetic absorption studies wereconducted in mice. The murine model has been very effective for smallscale inhalation studies [36]. The lung tissue concentration-timeprofiles following a single inhalation dose are shown in FIG. 8 whilethe corresponding pharmacokinetic parameters summarized in Table 5.

TABLE 5 In vivo pharmacokinetic parameters for the lung tissueconcentrations of the URF formulations. Formula- C_(max) T_(max) K_(el)T_(1/2) AUC₍₀₋₂₄₎ tions (μg/g) (hrs) (hrs⁻¹) (hrs) (μg · hr/g) URF-TAC10.86 ± 1.07 3 0.0346 20.02 111.19 ± 20.16 URF- 14.09 ± 1.50 2 0.033420.75 122.42 ± 6.19  TAC:LAC C_(max): maximum concentration T_(max):time to C_(max) K_(el): elimination rate constant T_(1/2): half-lifeAUC₍₀₋₂₄₎: area-under-the-curve between 0-24 hours

The C_(max) for URF-TAC:LAC was significantly higher (14.09 μg/g)compared to URF-TAC (10.86 μg/g) whereas T_(max) was significantly lower(p<0.05) for 2 hours. This could perhaps as a result of a greaterdissolved concentration, as seen in the vitro supersaturation results.However, no significant differences in AUC-values (0-24 hr) wereobserved between the two URF formulations (p>0.05). The resultsindicated that the amorphous nature of the particles affects the rate ofdrug absorption. TAC in the URF-TAC:LAC was eliminated according to abiphasic pattern with distribution phase and elimination phase. Thesimilar elimination pattern was also found in the URF-TAC The values ofIQ were not significantly different between the two URF-formulations(p>0.05). The decreasing TAC concentration in the lung for both URFformulations is a consequence of drug distribution and transport intothe systemic circulation, as well as particle elimination from the lung.It can be seen clearly that the transfer of nanostructured aggregates(either amorphous or crystalline) from the lung into systemiccirculation was likely in a sustained manner after 6 hr. The measuredlevels for both URF formulations at 48 hours were below the limit ofquantification of the assay (determined to bel μg/g).

The systemic in vivo pharmacokinetic of drug absorption from the lungswas investigated in mice. FIG. 9 shows a comparison of mean whole bloodconcentration-time profiles from each formulation, and the calculatedpharmacokinetic parameters following pulmonary administration arepresented in Table 6.

TABLE 6 In vivo pharmacokinetic parameters for the whole- bloodconcentrations of the URF formulations following the pulmonaryadministration. Formula- C_(max) T_(max) K_(el) T_(1/2) AUC₍₀₋₂₄₎ tion(ng/mL) (hrs) (hrs⁻¹) (hrs) (ng · hr/mL) URF-TAC 300.67 ± 27.04 3 0.1235.63 1324.35 ± 318.07 URF- 402.11 ± 35.99 2 0.115 6.02 1235.66 ± 65.86 TAC:LAC C_(max): maximum concentration T_(max): time to C_(max) K_(el):elimination rate constant T_(1/2): half-life AUC₍₀₋₂₄₎:area-under-the-curve between 0-24 hours

The whole blood concentration profile of each formulation has a similarabsorption pattern, for example, T_(max), compared to the lungconcentration profiles (FIG. 8). However, both URF formulationsdemonstrated substantially lower TAC concentrations in the blood thanwas seen in the lung tissue. The whole blood profiles followingpulmonary dosing of URF-TAC:LAC and URF-TAC had peak concentrations of402.11 ng/mL at 2 hr and 300.67 ng/mL at 3 hr, respectively, beforeconcentrations decreased. The AUC₍₀₋₂₄₎ (1235.66 ng·hr/mL) of theURF-TAC:LAC processed by URF is slightly lower than that of the URF-TAC(1324.35 ng·hr/mL), although there is no statistical difference(p>0.05). The levels of TAC decreased rapidly for URF-TAC:LAC with thelast time point with a detectable levels occurring at 24 h, whileURF-TAC declined in a similar but slower manner (no significantdifference in the IQ values (p>0.05)). Whole blood concentrations of TACwere below the limit of quantification for both formulations at 48hours. The systemic and lung concentrations observed after nebulizationof both URF formulations in mice suggest that a substantial lung andsystemic exposure to TAC can be achieved in either amorphousnanostructed aggregates or crystalline nanostructed aggregates producedby URF. The observation that either amorphous or crystalline particlesproduced high systemic concentrations may suggest that high surface areawas an important factor. High supersaturation, as a result of deliveringamorphous particles in URF-TAC:LAC, correlated with faster absorptionrates in both blood and lung tissue as compared to crystalline particlesin URF-TAC. Use of supersaturated state in the lungs has not beenpreviously studied yet. However, it has been demonstrated to enhancetransdermal and oral absorption of poorly soluble drugs [37-39]. The invivo data reported by Yamashita et al. [37] showed a high and extendedsystemic absorption of TAC following oral administration of amorphoussolid dispersions with HPMC in beagle dogs. In the Yamashita et al.study, the solid dispersion of TAC with HPMC was prepared by solventevaporation and was also shown to supersaturate in 0.1N HCl up to25-times in 2 hours, and this level was maintained for over 24 hours. Inour study, supersaturation of TAC from URF-TAC:LAC showed no effect onthe extent of drug absorption in both lung tissue and systemiccirculation. This can be explained by the fact that supersaturationoccurred over a short period of time for the absorption phase and thenTAC concentration was rapidly decreased in the elimination phase.

High surface area, nanostructured aggregates containing amorphous orcrystalline nanoparticles of TAC were produced by the URF process andshown to be effectively aerosolized in an aqueous dispersion bynebulization. Inclusion of lactose prevented crystallization of TAC andresulted in amorphous powder. URF-TAC:LAC (i.e., amorphousnanostructured aggregates) demonstrated the ability to supersaturate inSLF compared to the URF-TAC (i.e., crystalline nanostructuredaggregates). Dispersions of nebulized URF formulations exhibited highlung and systemic concentrations. The AUC₍₀₋₂₄₎ of the URF formulationswhich reflects the total amount of drug absorbed over the 24 h timeperiod was not significantly different (p>0.05) for either lung or bloodprofiles. The results indicate that high drug absorption in lung tissueand blood following pulmonary administration was primarily due to highsurface area of nanostructured aggregates from both formulations. Theability to achieve high solubility in the lungs translated to higherC_(max) and lower T_(max) values based on results of the in vivostudies. We have demonstrated that nanoparticles of TAC can besuccessfully delivered to the lungs without the use of polymers orsurfactants.

Example 1

A formulation of tacrolimus (TAC) was produced using TAC and lactose(LAC) in ratio 1:1. The TAC:LAC 1:1 formulation was prepared using theultra-rapid freezing (URF) process. The compositions were prepared bydissolving TAC and LAC at a 1:1 ratio and 0.75% solids in a 60/40mixture of acetonitrile and water. The solution of drug and excipientwas applied to the surface of a solid substrate, which is cooled using acryogenic substrate maintained at −50° C. The frozen compositions werethen collected and the solvent was removed by lyophilization using aVirTis Advantage Lyophilizer (VirTis Company, Inc. Gardiner, N.Y.). Thedried powders were stored at room temperature under vacuum.

Example 2

The composition in example 1 was characterized using X-ray powderdiffraction (XRD). The XRD patterns of the powders were analyzed using aPhilips 1710 x-ray diffractometer with a copper target and nickel filter(Philips Electronic Instruments, Mahwah, N.J.). Each sample was measuredfrom 5 to 45 2θ degrees using a step size of 0.05 2θ degrees and a dwelltime of one second. The composition is amorphous.

Example 3

The composition in example 1 was characterized using BET specificsurface area analysis. Specific surface area was measured using a Nova2000 v 6.11 instrument (Quantachrome Instruments, Boynton Beach, Fla.).A known weight of powder was added to a 12 mm Quantachrome bulb samplecell and degassed for a minimum of 3 hours. The data recorded were thenanalyzed according to BET theory using NOVA Enhanced Data ReductionSoftware v. 2.13. The results showed that the composition has a BETspecific surface area of 25.9 m²/g, compared to 0.53 m²/g forunprocessed TAC.

Example 4

The composition in example 1 was characterized using scanning electronmicroscopy (SEM) in order to visualize the morphology of the particlesproduced. A Hitachi S-4500 field emission scanning electron microscope(Hitachi High-Technologies Corp., Tokyo, Japan) was used to obtain SEMmicrographs of the powder samples. Samples were mounted on conductivetape and sputter coated using a K575 sputter coater (Emitech Products,Inc. Houston, Tex.) with gold/palladium for 30 sec. An accelerationvoltage of 5-15 kV was used to view the images. The results of the SEMcharacterization of the powders showed highly porous nanostructuredaggregates of TAC/LAC. The micrograph at high magnification revealedthat the aggregates were composed of interconnected nanoparticles with adiameter of approximately 100-200 nm.

Example 5

The composition in example 1 was tested for its dissolutioncharacteristics at sink conditions (defined here as 59% of equilibriumsolubility in the dissolution medium) below the equilibrium solubilityof TAC. Dissolution testing at conditions below equilibrium solubilitywas performed on the TAC:LAC formulation using a United StatesPharmacopoeia (USP) 27 Type 2 dissolution apparatus (Vankel VK6010Dissolution Tester with a Vanderkamp VK650A heater/circulator, Varian,Inc. Palo Alto, Calif.). Powder samples (0.4 mg TAC) equivalent toapproximately 59% of the equilibrium solubility (6.8 μg/mL) were addedto 100 mL of modified simulated lung fluid (SLF) with 0.02% DPPC as thedissolution medium. The dissolution medium was maintained at 37.0±0.2°C. and stirred at a constant rate of 100 RPM. Samples (1 mL) werewithdrawn at 10, 20, 30, 60, and 120 minute time points, filtered usinga 0.45 μm GHP Acrodisc filter (VWR, Inc. Westchester, Pa.), and analyzedusing a Shimadzu LC-10 liquid chromatograph (Shimadzu Corporation,Kyoto, Japan) equipped with an Altech ODS-2, 5 μm C18 column (AltechAssociates, Deerfield, Ill.). The mobile phase consisted of a 70:30(v/v) ACN:water mixture, using a flow rate of 1 mL/min. The maximumabsorbance was measured at wavelength λ=214 nm. The results of thedissolution testing below equilibrium solubility are shown in the Table7:

Time (min) % Tacrolimus Dissolved 0 0 10 41 20 59 30 73 60 82 120 94

Example 6

The composition in example 1 was tested for its dissolutioncharacteristics under supersaturated conditions. Supersaturateddissolution profiles were generated according to the method described inexample 5, using the small volume dissolution apparatus equipped with apaddle stirring mechanism. The drug formulation was weighed tocorrespond to approximately 15-times the aqueous crystalline solubilityof TAC in 100 mL of the modified simulated lung fluid with 0.02% DPPC.Paddle speed and bath temperature were maintained at 100 RPM and 37.0°C., respectively. An aliquot (1 mL) was removed from the small volumevessel at 10, 20, 30, and 60 minutes, then at 2, 4, and 24 hours. Eachaliquot was filtered through a 0.2 μm nylon filter, and a 0.5 mL aliquotof each filtered solution was immediately mixed with 1 mL ofacetonitrile (to ensure no re-crystallization of drug previouslydissolved at 37° C.). The samples were analyzed for TAC concentrationusing the same HPLC procedure described in example 5. The results forthe supersaturated dissolution tests are shown below, Table 8.

TABLE 8 Time (min) Relative TAC Conc. (C/Ceq) 0 0 10 5.3 20 6.9 30 9.760 10.6 120 6.7 240 3.1 1440 1.1

Example 7

The composition in example 1 was tested for its performance in vivo inthe mouse, using pulmonary administration of the composition inexample 1. Pulmonary dosing of the formulation was performed in healthyICR mice (Harlan Sprague Dawley, Indianapolis, Ind.). The study protocolwas approved by the Institutional Animal Care and Use Committee (IACUC)at the University of Texas at Austin, and all animals were maintained inaccordance with the American Association for Accreditation of LaboratoryAnimal Care. Mice were acclimated in the restraint tubes (Battelle,Columbus, Ohio) for 10-15 min/day for 2 days prior to dosing. A smallanimal dosing apparatus for inhalation was used to dose the mice for thestudy. The dosing apparatus was designed to hold up to 4 mice per dosingtime point. The dosing apparatus consists of a small volume hollow tubewith dimensions of 20×4.5 cm with four 1.75 cm adapter holes drilled at7 cm intervals, in order to accept rodent restraint tubes from theBattelle toxicology testing unit. The composition of Example 1 wasre-dispersed in water (10 mg/mL) followed by sonication for 1 min priorto dosing to prepare the nebulization suspension. Nebulization of 3 mLof prepared suspension was conducted using an Aeroneb® Professionalmicropump nebulizer for 10 min. After pulmonary dosing, the mice wereremoved from the dosing apparatus and rested for 15 min. Two mice weresacrificed at each time point by CO₂ narcosis (0.5, 1, 2, 3, 6, 12, 24,and 48 hours). Whole blood (1 mL aliquots) was obtained via cardiacpuncture and analyzed using a PRO-Trac II FK 506 ELISA, following theprocedure detailed in the PRO-Trac II FK 506 ELISA assay kit literature(Diasorin, Inc. Stillwater, Okla.). In addition, necropsy was performedon each mouse to extract lung tissue. Samples were stored at −20° C.until assayed. TAC concentration in the lung tissue was determined byusing the HPLC method described in example 5. The results for the bloodand lung tissue TAC concentrations are shown in Tables 9 and 10:

TABLE 9 TAC Whole Blood Concentrations following Pulmonary Dosing: Mouse1 Mouse 2 Average Time TAC Conc. TAC Conc. TAC Conc. (hr) (ng/mL blood)(ng/mL blood) (ng/mL blood) 0 0 0 0 0.5 33.68 16.22 24.95, SD = 8.73 1254.97 281.42 268.20, SD = 13.22 2 427.56 376.65 402.11, SD = 25.45 3155.70 86.72 121.21, SD = 31.50 6 49.56 86.65  68.11, SD = 18.55 1226.25 11.13 18.68, SD = 7.56 24 5.88 8.05  6.97, SD = 1.09 48 N/A N/AN/A

TABLE 10 TAC Lung Tissue Concentrations following PulmonaryAdministration: Mouse 1 Mouse 2 Average Time TAC Conc. TAC Conc. TACConc. (hr) (μg/g lung weight) (μg/g lung weight) (μg/g lung weight) 0 00 0 0.5 7.31 9.05 8.06, SD = 0.99 1 11.41 12.27 10.10, SD = 2.09  213.59 14.14 14.09, SD = 1.50  3 6.73 8.61 8.22, SD = 1.18 6 4.46 4.195.02, SD = 1.00 12 5.95 6.11 4.80, SD = 1.42 24 3.29 2.93 3.51, SD =0.59 48 N/A N/A N/A

Example 8

A formulation of tacrolimus (TAC) was produced using TAC and glucose(GLU) in ratio 1:1. The TAC:GLU 1:1 formulation was prepared using theultra-rapid freezing (URF) process. The composition was prepared bydissolving TAC and GLU at a 1:1 ratio and 0.75% solids in a 60/40mixture of acetonitrile and water. The solution of drug and excipientwas applied to the surface of a solid substrate, which is cooled using acryogenic substrate maintained at −50° C. The frozen compositions werethen collected and the solvent was removed by lyophilization using aVirTis Advantage Lyophilizer (VirTis Company, Inc. Gardiner, N.Y.). Thedried powders were stored at room temperature under vacuum. The resultof XRD characterization (following the procedure in example 2) is thatthe formulation is amorphous. The result of SEM (following the procedurein example 4) is that the morphology is nanostructured aggregates withsmall primary particles consisting of TAC and GLU with primary particlesizes of about 100-300 nm.

Example 9

A formulation of tacrolimus (TAC) was produced using TAC and mannitol(MAN) in ratio 1:1. The TAC:MAN 1:1 formulation was prepared using theultra-rapid freezing (URF) process. The composition was prepared bydissolving TAC and MAN at a 1:1 ratio and 0.75% solids in a 60/40mixture of acetonitrile and water. The solution of drug and excipientwas applied to the surface of a solid substrate, which is cooled using acryogenic substrate maintained at −50° C. The frozen compositions werethen collected and the solvent was removed by lyophilization using aVirTis Advantage Lyophilizer (VirTis Company, Inc. Gardiner, N.Y.). Thedried powders were stored at room temperature under vacuum. The resultof XRD characterization (following the procedure in example 2) is thatthe composition is amorphous. The result of SEM (following the procedurein example 4) is that the morphology is nanostructured aggregates withsmall primary particles consisting of TAC and MAN with primary particlesizes of about 100-200 nm.

Example 10

A formulation of tacrolimus (TAC) was produced using TAC and inulin(INL) in ratio 1:1. The TAC:INL 1:1 formulation was prepared using theultra-rapid freezing (URF) process. The composition was prepared bydissolving TAC and INL at a 1:1 ratio and 0.75% solids in a 60/40mixture of acetonitrile and water. The solution of drug and excipientwas applied to the surface of a solid substrate, which is cooled using acryogenic substrate maintained at −50° C. The frozen compositions werethen collected and the solvent was removed by lyophilization using aVirTis Advantage Lyophilizer (VirTis Company, Inc. Gardiner, N.Y.). Thedried powders were stored at room temperature under vacuum. The resultof XRD characterization (following the procedure in example 2) is thatthe formulation is amorphous. The result of SEM visualization (followingthe procedure in example 4) is that the formulation's morphology isnanostructured aggregates with small primary particles consisting of TACand INL with primary particle sizes of about 100-200 nm.

Example 11

In order to evaluate the rodent dosing apparatus used in these studies,an in vitro and in vivo study of the apparatus was conducted usingitraconazole (ITZ). An animal dosing inhalation apparatus wasconstructed, consisting of a hollow tube (20×4.5 cm, nominal wallthickness of 0.4 cm) with four 1.75 cm adapter holes drilled at 7 cmintervals (2 holes on each side). The adapter holes were constructed toaccept rodent restraint tubes from the Battelle toxicology testing unit.A ITZ colloidal suspension was nebulized into the apparatus for 5 minusing a micro-pump nebulizer. Atomized droplets containing ITZ weredriven into the chamber at a flow rate of 1 mL/min. ITZ concentrationswere measured in vitro at the 4 adapter ports, and in vivo from thelungs of male outbred ICR mice in the appropriate mice restraining tubesat the adapter ports. The in vitro results showed that ITZconcentrations (S.D.) were 3.35 (0.75) μg/mL at the adapter portsfollowing 5 min nebulization. In vivo results showed that lungconcentrations of ITZ were 32 (3.0) μg/g wet lung weight (n=8). This wasfound to be three times higher than had previously been determined usinga restraint-free whole body exposure unit in the same strain of mousewith double the exposure time. High concentrations of ITZ are achievedin the rodent lung with low variability. A commercially availablenebulizer can be used for short dosing periods that negate the need touse invasive and variable dosing techniques. The data for the in vivomouse study is Table 11:

TABLE 11 ITZ Conc. in Lung Tissue. Mouse ITZ Conc. in Lung Tissue (μg/g)Mouse 1 13.5 Mouse 2 15.9 Mouse 3 18.6 Mouse 4 19.2 Mouse 5 22.7 Mouse 621.6 Mouse 7 20.2 Mouse 8 18.3 Average 18.7, SD = 3.0

Example 12

The composition in example 1 was tested for its performance in vivo inthe mouse model after multiple dosing. The dosing apparatus described inexample 7 was used. Re-dispersion and dosing concentration described inexample 7 was also used; however, dosing occurred once daily. Two groupsof four mice were sacrificed by isoflurane inhalation after multipledosing. One group of four received 6 doses, the other received 13 doses.Animals were sacrificed 24 hours after the last dose was administeredand trough blood samples were taken. Whole blood and lung tissue sampleswere extracted and assayed as described in example 7. The results forthe blood and lung tissue TAC concentrations are shown in Table 12 and13.

TABLE 12 TAC Whole Blood Concentrations following Daily Pulmonary Dosingin Mice Avg. Blood Avg. Amount of Day of Volume TAC Conc. TAC in BloodSacrifice (mL) (ng/mL) (μg) 7 2.4 2.39 0.0057 14 2.4 2.649 0.0064

TABLE 13 TAC Lung Concentrations following Daily Pulmonary Dosing inMice Avg. Lung Avg. Amount of Day of Weight TAC Conc. TAC in LungSacrifice (g) (μg/g) (μg) 7 0.2205 7.19 1.585 14 0.2193 6.735 1.477

Example 13

Lung tissue from the study conducted in example 12 was subjected tohistological examination. Lungs were inflated with 10% formalin solutionafter sacrifice, tied at the trachea, and extracted. Sections of thelungs were taken, stained, and embedded in paraffin wax. Along with theactive dosing groups described in example 12, a control group dosed withlactose solution for 6 and 13 days was evaluated. No evidence of tissuedamage was observed in either case. Images from microscopic evaluationare shown in FIG. 10A-10D.

Example 14

The composition in example 1 was tested for its performance in vivo inthe rat model at a lowered dose in comparison to the dose in example 7.Pulmonary dosing of the formulation was performed in healthy SpragueDawley rats (Harlan, Indianapolis Ind.). The study protocol was approvedby the Institutional Animal Care and Use Committee (IACUC) at theUniversity of Texas at Austin, and all animals were maintained inaccordance with the American Association for Accreditation of LaboratoryAnimal Care. Rats were acclimated in the restraint tubes (Battelle,Columbus, Ohio) for 10-15 min/day for 2 days prior to dosing. A smallanimal dosing apparatus for inhalation was used to dose the rats for thestudy. The dosing apparatus was designed to hold up to 4 rats per dosingtime point. The dosing apparatus consists of a small volume hollow tubewith dimensions of 2×4.5 cm with four 1.75 cm adapter holes drilled at 7cm intervals and staggered on either side, in order to accept rodentrestraint tubes from the Battelle toxicology testing unit. Thecomposition of Example 1 was re-dispersed in water (1.1 mg/mL) followedby sonication for 1 min prior to dosing to prepare the nebulizationsuspension. Nebulization of 3 mL of prepared suspension was conductedusing an Aeroneb® Professional micropump nebulizer for 10 min. Afterpulmonary dosing, the rats were removed from the dosing apparatus,rested for 1 hour, then euthanized by CO2 narcosis. Whole blood aliquotswere extracted and assayed as described in example 7. The results forthe whole blood TAC concentrations are shown in Table 14

TABLE 14 TAC Whole Blood Concentrations following PulmonaryAdministration at a Lowered Dose in Rats TAC Conc. in Blood Rat (ng/mL)Rat 1 7.20 Rat 2 1.53 Rat 3 4.42 Rat 4 2.50 Average 3.91, SD = 2.5

Example 15

The composition in example 1 was tested for its performance in vivo inthe rat after multiple dosing. The dosing apparatus described in example14 was used. Re-dispersion and dosing concentration described in example14 was also used; however, dosing occurred once daily. Eight rats weresacrificed by isoflurane inhalation after 21 doses. Animals weresacrificed 24 hours after the last dose was administered and troughblood samples were taken. Whole blood samples were assayed as describedin example 14. The results for the whole blood TAC concentrations areshown in Table 15.

TABLE 15 TAC Whole Blood Concentrations following 21 Days ContinuousPulmonary Administration at a Lowered Dose in Rats TAC Conc in Blood Rat(ng/mL) Rat 1 2.2096 Rat 2 1.8193 Rat 3 1.4874 Rat 4 2.7917 Rat 5 1.4187Rat 6 1.4075 Rat 7 1.4874 Rat 8 1.3742 Average 1.75, SD = 0.5

Example 16

The composition in example 1 was tested for its performance in vivo in arat lung transplant model at the lowered dose used in example 14.Pulmonary dosing of the formulation was performed in healthy, lungtransplanted Sprague Dawley rats (Harlan, Indianapolis Ind.). Surgerywas performed to replace the left lung with a healthy left lung from thesame species. Rats were given at least 7 days before dose wasadministered. The study protocol was approved by the InstitutionalAnimal Care and Use Committee (IACUC) at the University of Texas HealthScience Center in San Antonio, and all animals were maintained inaccordance with the American Association for Accreditation of LaboratoryAnimal Care. Dosing was conducted as detailed in example 14; however,euthanasia was performed by tissue necropsy after isoflurane anesthesia.Whole blood aliquots were extracted from 3 transplanted rats for twotime points and assayed as described in example 7. The results for thewhole blood TAC concentrations at 6 and 12 hrs were 2.97±0.3 and2.55±0.3 ng/mL, respectively. Right and left lung tissue samples werealso harvested and analyzed for TAC from 3 transplanted rats at two timepoints content by liquid chromatography/mass spectrometry (LC/MS).Briefly, lung tissue was homogenized and proteins were precipitated toseparate the analyte. Samples were spiked with an internal standard toassess and correct for extraction efficiency. The results for left(transplanted) lung TAC at 6 and 12 hrs were 319.8±80 and 160.4±46 ng/g,respectively. The results for right lung TAC concentration at 6 and 12hrs were 125.0±5 and 62.6.4±17 ng/g, respectively.

Example 17

The composition in example 1 was tested for its performance in vivo in arat lung transplant model described in example 16 at the lowered doseused in example 14. After sacrifice, lungs were extracted and sectionedinto proximal airway and distal airway portions. These sections wereanalyzed for percent of total lung TAC according to mass by LC/MSaccording to example 16. The results for right proximal, right distal,left proximal, and left distal in 3 lung transplanted rats at the sixhour time point were 4.0±6%, 49.3±4%, 33.6±4%, and 13.1±5% total TACdeposited, respectively. In a single non-transplanted rat in the samestudy, results for right proximal, right distal, left proximal, and leftdistal at the six hour time point were 2.6%, 49.0%, 25.1%, and 23.3%total TAC deposited, respectively.

Example 18

The composition in example 1 was tested for its in vitro performance inmixed lymphocyte culture (MLC) immune response analysis. This test isinitiated by culturing bone marrow cells from a transplant host withcells from a transplant donor. Lymphocyte proliferation in this culturewas assessed for histocompatibility without tacrolimus, after theaddition of Prograf® dissolved in ethanol, and after the addition ofTAC:LAC composition dispersed in water. Percent inhibition wasdetermined relative to the culture lymphocyte count without the presenceof tacrolimus. An average inhibition was calculated after fouriterations. It was found that at equivalent doses, Prograf® dissolved inethanol inhibited lymphocyte proliferation by 45%, while TAC:LACdispersed in water inhibited proliferation by 86%.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1-44. (canceled)
 45. A pharmaceutical composition comprisingimmunosuppressive drug-containing nanoparticles, the nanoparticlescomprising an amorphous immunosuppressive drug and a carbohydrate, saidnanoparticles prepared by a process comprising mixing the amorphousimmunosuppressive drug with the carbohydrate; and ultra-rapid freezingthe drug and carbohydrate into a high potency amorphous nanoparticle byultra-rapid freezing on a solid substrate.
 46. The composition of claim45, wherein the nanoparticles are free of polymeric surfactants.
 47. Thecomposition of claim 45, wherein the carbohydrate is mannitol,raffinose, lactose, maltodextrin, trehalose or combinations thereof. 48.The composition of claim 47, wherein the carbohydrate is lactose. 49.The composition of claim 45, wherein the nanoparticles are free ofethanol and propylene glycol.
 50. The composition of claim 45, whereinthe nanoparticles are further defined as nanostructured particles. 51.The composition of claim 45, further defined as a dispersion or powderadapted for nebulization or inhalation.
 52. The composition of claim 49,comprised in a nebulizer, an air-jet nebulizer, an ultrasonic nebulizer,a metered dose inhaler, a dry powder inhalation device or a micro-pumpnebulizer.
 53. The composition of claim 45, wherein theimmunosuppressive drug is tacrolimus.
 54. The composition of claim 45,wherein the immunosuppressive drug is cyclosporine.
 55. The compositionof claim 45, wherein the solid substrate is a cryogenic solid substrate.56. The composition of claim 45, wherein the solid substrate is cooledsubsequent to the application of the drug and carbohydrate thereon. 57.The composition of claim 45, wherein the solid substrate is cooled priorto the application of the drug and carbohydrate thereon.
 58. A method oftreating a pulmonary disease in a subject, the method comprisingobtaining a pharmaceutical composition in accordance with claim 45, anddelivering the immunosuppressive drug-containing nanoparticles to thesubject by pulmonary delivery to treat one or more pulmonary diseases inan amount effective to treat the pulmonary disease.
 59. The method ofclaim 58, wherein the pulmonary disease is pulmonary fibrosis.
 60. Themethod of claim 58, wherein the pulmonary disease is bronchiolar asthma.61. The method of claim 58, wherein the pulmonary disease is graftrejection.
 62. The method of claim 58, wherein the pharmaceuticalcomposition is delivered to the lungs of the subject by nebulization,metered dose inhalation or dry powder inhalation.